In the United States, millions of people are affected by tissue loss every year. Current treatments include tissue transfer from a healthy site in the same or another individual, use of medical devices to support the function of the lost tissue, or pharmacologic supplementation of the metabolic products of the lost tissue. Problems with these current treatments include potential tissue complications and imperfect matches including the possible dependence on immunosuppressants, limited durability of the mechanical devices, and the inconvenience and complexity of pharmacologic supplementation. Current approaches for developing living tissue substitutes make use of a “scaffold” that serves as a physical support and template for cell attachment and tissue development. These scaffolds are ideally designed to resemble, both in structure and composition, the extracellular matrix that the cells are exposed to in vivo, in order to simulate the in vivo conditions. An early and widely used natural scaffold is made of the extracellular matrix protein collagen, while more recently, mechanically stronger artificial scaffolds made of substances such as poly-glycolic acid (PGA) and poly-lactic acid (PLA) have been used.
Some cell-scaffold compositions have multiple layers of biocompatible materials including extracellular matrix materials such as collagen, fibril-forming collagen, Matrix Gla protein, osteocalcin, or other biocompatible materials including marine coral, coralline hydroxyapatite ceramic, and mixtures thereof, and some such scaffolds have been seeded with cells, and then placed within a bioreactor having a means for mechanically stimulating the cells at distinct frequencies (see U.S. Patent Application No. 0040005297 to P. R. Connelly et al., filed Jul. 8, 2002, published Jan. 8, 2004).
In addition, living tissue equivalents (LTEs), notably cell-seeded collagen and fibrin gels, have been used extensively as in vitro wound-healing models as well as systems for studying tissue remodeling. More recently, LTEs have begun to gain considerable attention as replacements for lost or damaged connective tissue (e.g., Apligraf™ from Organogenesis, Inc.). LTEs have several advantages over synthetic alternatives including being a natural cell substrate, allowing cellularity to be achieved directly, and being conducive to cell spreading and extracellular matrix (ECM) formation. LTEs are made by mixing cells with a soluble biopolymer solution (e.g., collagen, fibrin, and/or proteoglycans). The cells invade, rearrange and partially degrade the biopolymer scaffold over the next few days as well as synthesize new proteins throughout the culture period. However, LTEs generally lack the physical properties necessary to resist in vivo mechanical forces, and are not true “living tissues”.
Over the last two decades, LTEs that are completely cell-derived have been developed. However, to date they have been very thin and taken a long time to grow, generally on the order of months, whereas collagen gels and fibrin gels can be developed in only a few days. There is a need for completely biological cell-derived LTEs, and living scaffolds for use in wound repair and tissue regeneration in vitro and in vivo.